1. Field of the Invention
The present invention relates generally to delivery and retention of active agents at a targeted site using compositions comprising ligand and anti-ligand conjugates. More particularly, certain embodiments concern methods, compounds, compositions and kits useful for targeted delivery and retention of agents at specific lymph nodes by administration of a composition comprising a ligand-colloid moiety and a composition comprising an anti-ligand. In certain embodiments the ligand is one member of the biotin/avidin pair and the anti-ligand is the other member of the biotin/avidin pair.
2. Description of Related Art
Avidins are a family of proteins functionally defined by their high affinity for binding biotin. Avidins are small oligomeric proteins made up of four identical subunits, each with a single binding site for biotin. Avidins include proteins (1) present in the eggs of amphibians, reptiles, and birds, and known as avidin, and (2) produced by Streptomyces avidinii and known as streptavidin. Streptavidin is similar to avidin in its binding properties, but has lower non-specific tissue binding, and therefore often is used in place of avidin. However, the immunogenicity of streptavidin is a major drawback to its use. Paganelli et al., Eur J Nucl Med 24(3):350–351, 1997. Consequently, modified avidins with lower immunogenicity than that of streptavidin have been developed. Chinol et al., Brit J Cancer 78(2): 189–197, 1998; Goldenberg, U.S. Pat. No. 5,698,405, incorporated herein in their entirety by reference. Chinol et al. found that conjugating avidin to monomethoxypolyethyleneglycol 5000 (mPEG), in particular an average of 7 mPEG chains per avidin molecule, produced a compound with substantially reduced immunogenicity and low cross-reactivity with native avidin. Goldenberg reduced the immunogenicity of avidin by coupling it with a carbohydrate polymer or polyol groups. As used hereafter, “avidin” includes all of the above proteins.
Biotin is a natural water-soluble vitamin found in every living cell. Several derivatives of biotin are commercially available. Due to the extremely high affinity of avidins for biotin, the biotin/avidin system has been used for targeting, detecting, and treating tumors. The tetravalency of avidin for biotin permits amplification when avidin binds to biotin.
The biotin/avidin system has been used primarily in conjunction with antibodies targeting specific tissues or lesions, as described, for example, in Hnatowich et al., Nucl Med Biol 20:189–195, 1993; Ogihara-Umeda et al., Cancer Detection and Prevention 21(6):490–496, 1997; Lanza, U.S. Pat. No. 5,690,907; Goldenberg, U.S. Pat. Nos. 5,736,119 and 5,776,094; and Griffiths, U.S. Pat. No. 5,482,698, incorporated herein in their entirety by reference. Antibodies directed against different determinants associated with pathologic or normal cell type or pathogenic organisms, wherein avidin or biotin is conjugated to the targeting antibody, have been used to target tissues and lesions, as described in, for example, Griffiths, U.S. Pat. No. 5,482,698 and Goldenberg, U.S. Pat. No. 5,776,094. In such applications, the biotin/avidin system has been used as a means for delivering a detection agent to a site previously targeted by antibody and for clearing excess targeting antibody from the circulation in order to increase the target:background ratio.
Two basic approaches for targeting specific sites in a subject with biotin/avidin systems have been used in mammalian subjects. In a 2-step procedure, a targeting antibody is conjugated with either avidin or biotin and then is injected into a subject, thus localizing the avidin or biotin at a site of interest. Thereafter, either biotin or avidin (depending on which was coupled to the targeting antibody) bearing an active agent is injected and is localized at the site of the primary antibody by binding to avidin or biotin respectively. Timing of the second injection after the first one is very critical. Injecting the active agent-avidin (or biotin) too early will increase the avidin/biotin conjugates in the bloodstream and nontargeted tissues, while injecting too late may decrease the amount targeted to the desired site because of reduced retention of the primary antibody at the tumor.
Incomplete clearance of unbound antibody from the blood circulation can obscure detection of the target site. In the second method for targeting specific sites with the biotin/avidin system, blood background is reduced by injecting biotinylated antibodies followed three days later by cold (unlabeled) avidin. The resultant circulating biotinylated antibody-avidin complexes are sequestered from the blood by the liver. Radioactive biotin is then injected and binds to the antibody-biotin-avidin complexes already localized at the targeted site.
A drawback of both of the above approaches for targeting tumors is that they require that a subject be available to undergo multiple procedures over a prolonged time, generally a few days.
Essential to known uses of the biotin/avidin system for detection and therapy of specific sites in the body, such as tumors, is the element of “pretargeting,” as described, for example, in Hnatowich et al, J Nucl Med 28(8):1294–1302, 1987; Paganelli et al., Nucl Med Commun 12(3):211–234, 1991; Paganelli et al., Eur J Nucl Med 19(5):322–329, 1992; Griffiths, U.S. Pat. No. 5,482,698, Goodwin et al., U.S. Pat. No. 4,863,713; Goodwin et al., J Nucl Med 29(2):226–234, 1988; Oehr et al., J Nucl Med 29:728, 1988; Klibanov et al., J Nucl Med 29(12):1951–1956, 1988; Sinitsyn et al., J Nucl Med 30(1):66–69, 1989; Kalofonos et al., J Nucl Med 31(11):1791–1796, 1990; Schechter et al., Int J Cancer 48(2):167–172, 1991; Paganelli et al., Cancer Res 51(21):5960–5966, 1991; Stickney et al., Cancer Res 51(24):6650–6655, 1991; and Yuan et al., Cancer Res 51(12):3119–3130, 1991; all incorporated herein in their entirety by reference. Pretargeting makes use of the biotin/avidin system to eliminate detection and therapeutic agents from sites other than the targeted sites. Pretargeting does not make use of the biotin/avidin system to directly target sites, as occurs with the present invention.
In pretargeting, a targeting compound comprising a targeting moiety such as antibody conjugated to one member of the biotin/avidin pair is administered to a subject. A portion of the targeting compound distributes to the targeted site and is bound there. The significant portion of the targeting compound may be retained in the blood pool. After a predetermined time period, typically a few hours to two to three days, a compound comprising the member of the biotin/avidin system that is not conjugated to a targeting moiety is administered and binds with the biotin or avidin of the circulating targeting compound. A detection or therapeutic agent is incorporated with either the targeting compound, or the subsequently injected avidin or biotin compound, or both, prior to administration. When bound, the newly formed compound comprising targeting moiety-biotin-avidin-detection/therapeutic agent is removed from circulation by the reticuloendothelial system, thus clearing the compound from those portions of the body that are not targeted and lowering the background level of the compound. This increases the target-to-background ratio and increases the ability to detect the targeted site High background levels of a detection agent have long been recognized as a major impediment to achieving the high target:background ratios desirable for detection of lesions. Furthermore, high background levels of therapeutic agents can lead to toxicity and restrict the therapeutic agents and dosages that can be safely used for therapy.
Liposomes have been described as potential agents for targeted delivery of diagnostic or therapeutic agents to a wide range of organ systems and diseases. Such targeting is due primarily to a physical feature of the liposome, such as size, charge, and lipid compound, and is not due to specific site-directed targeting. Phillips et al., Handbook of Targeted Delivery of Imaging Agents, CRC Press, 149–173, 1995, incorporated herein in its entirety by reference. Coupling macrophage-specific ligands to the surface of liposomes increases liposome drainage from an interstitial injection site and enhances their localization in regional lymph nodes. Moghimi et al., Prog Biophys Molec Biol 65:221–249, 1996, incorporated herein in its entirety by reference.
High background levels of radiolabeled liposomes following intravenous injection can impede the high target:background ratios needed for detection of a lesion. Ogihara-Umeda et al., Cancer Detection and Prevention 21(6):490–496, 1997, demonstrated that radiolabeled liposomes coated with biotin accumulated in a tumor, but that target:background ratios were improved following an intravenous injection of avidin. The avidin was used to remove the remaining radiolabeled biotin-liposomes in the blood circulation by binding together to form crosslinked complexes which were cleared by the liver.
The lymphatic system plays important roles in transporting body fluids and particulate materials from the body's periphery to the thoracic duct, returning large proteins and lymphocytes to the blood circulation from the tissue fluid, and transporting the products of fat digestion in the gastrointestinal tract (chylomicrons) into the blood circulation. The properties of the lymphatic system have been reviewed in detail by Yoffrey et al., Lymphatics, lymph and the lymphomyeloid complex, Academic Press, London, 1970, incorporated herein in its entirety by reference. The lymphatic system also plays an important role in the spread of a variety of disease processes, as described, for example, by Papisov et al., Crit Rev Ther Drug Carrier Syst 13(1&2):57–84, 1996, incorporated herein in its entirety by reference. Lymphatic dissemination of disease processes allows the spread of disease to regional lymph nodes, and even further. For example, malignant cells can enter the lymphatic system and become captured by lymph nodes; the lymph nodes consequently serve as foci of residual metastatic cancer, with potential tumor recurrence even after treatment of the primary tumor. Weiss et al., Lymphatic System Metastases, Hall, Boston, 1988. The lymph can also be involved in the spread of tumors to other organs. Consequently, there is considerable need for a method of determining and examining lymph nodes involved in the dissemination of disease processes and in delivering therapeutic agents to lymph nodes. This subject has been reviewed extensively by Hader et al;, AORN J 68:572–588, 1998, and Tanabe et al., Advances in Surgery 31:79–103, 1998, incorporated herein in their entirety by reference.
Any material which transits from the interstitial space to the intralymphatic space will move to a series of lymph nodes, the regional lymph nodes, that drain the lymph toward the thoracic duct. The first such lymph node encountered is the primary lymph node. Lymph from the primary lymph node will pass to subsequent lymph nodes, called secondary lymph nodes.
The sentinel lymph node is the first lymph node encountered by a metastasizing tumor cell after it has entered the lymphatic system. The importance of the sentinel lymph node lies in the fact that metastasizing tumor cells are recognized by the immune system and stopped there. Many times, these tumor cells are destroyed by the immune cells located in the sentinel lymph node. However, tumor cells can survive, creating a foci of metastatic disease in the sentinel lymph node.
If tumor cells have metastasized to other locations in the body, malignant tumor cells will be found in the sentinel lymph node 99% of the time. On the other hand, if no tumor cells are found in the sentinel lymph node after close pathological examination, it is very unlikely that the cancer will reoccur after the primary tumor has been removed For these reasons, it is very important to locate the sentinel lymph node and, if necessary, target treatment specifically to it.
Locating the sentinel lymph node in a mammalian subject is not always easily accomplished. Lymph nodes tend to blend in with the rest of the body tissue. In addition, it is not readily apparent which of numerous lymph nodes identified drains a disease locus, such as a tumor bed. Furthermore, occasionally a disease locus can drain to more than one area, thus there can be multiple sentinel lymph nodes. The present invention addresses the need to identify sentinel lymph nodes.
Previous methods for directing detection or therapeutic agents to lymph nodes have employed colloids, especially liposomes, to passively target lymphatic tissue, as described, for example, in Moghimi et al., Prog Biophys Molec Biol 65:221–249, 1996, Davis, U.S. Pat. No 5,792,475; Wolf, in Handbook of Targeted Delivery of Imaging Agents, Chap. 21, 366–384, CRC Press, 1995; Papisov et al., Crit Rev Ther Drug Carrier Syst 13(1&2):57–84, 1996, all of which are incorporated herein in their entirety by reference. Interstitial administration of colloids such as liposomes results in accumulation of such colloids in lymphatic tissue, and is described, for example, in Oussoren et al., Biochim Biophys Acta 1328:261–272, 1997, incorporated herein in its entirety by reference. Efficient accumulation of macromolecular carriers in lymph nodes after intralymphatic injection of macromolecules has also been described. See, for example, Papisov, Crit Rev Ther Drug Carrier Syst 13(1&2):57–84, 1996.
Methods for detecting sentinel lymph nodes have been described. In one method microcolloidal particles labeled with a radioisotope are administered interstitially proximal to the tumor site and scintigraphic scans or radio-guided probes are used to locate the site(s) of maximum radioactivity. This method is described, for example, in Van der Veen et al., Br J Surg 81(12):1769–1770, 1994; Krag et al., Surg Oncol 2:335–339, 1993; Veronesi et al., Lancet 349(9069):1864–1867, 1997, all of which are incorporated herein in their entirety by reference. In another method, vital blue dye is injected peri-tumor, as described, for example, in Morton et al., Surg Oncol Clin N Am 1:247–59, 1992 and Arch Surg 127(4):392–399, 1992, incorporated herein in their entirety by reference. An intraoperative method for detecting sentinel lymph nodes using both radiolabeled colloid and vital blue dye has also been described, for example, in Cox et al., Ann Surg 227(5):645–653, 1998, incorporated herein in its entirety by reference. In this method radiolabeled colloid is injected around the periphery of a tumor site one to six hours prior to an operative procedure. Immediately before the operative procedure, vital blue dye is injected peri-tumor. The vital blue dye stains afferent lymphatic channels to aid in visual localization of the sentinel lymph node. Prior to skin incision, a hand-held gamma-detection probe is used to localize the sentinel lymph node. After incision, the gamma probe is used to guide localization of the sentinel lymph node. See, for example, Kotz, J Nucl Med 39(12):13N–21N, 1998; Krag et al., N Engl J Med 339(14):941–946, 1998; Reintgen, J Nucl Med 39(12):22N–36N, 1998, all of which are incorporated herein in their entirety by reference.
Injection of a radiolabeled colloid to detect the sentinel lymph node by a radioactive probe has been used in conjunction with injection of blue dye to visualize the sentinel lymph nodes. A problem with this approach is that the radiolabeled colloid and the blue dye do not move through the lymphatic system at the same rate. The blue dye is absorbed rapidly from its site of injection and readily passes through lymph nodes. In contrast, the radiolabeled colloid is absorbed more slowly, takes time to accumulate in the lymph node, and does not significantly pass through the first lymph node encountered to other lymph nodes. Consequently, the timing of the localizing surgical procedure is difficult because simultaneous accumulation of blue dye and radiolabeled colloid at the sentinel node requires very different injection times for the blue dye and radiolabeled colloid.
A targeting system for the delivery of diagnostic and therapeutic agents to the lymphatic system should have the following characteristics: (i) spread well from the injection site, (ii) provide good uptake in primary lymph node(s) if sentinel lymph node is desired, and (iii) provide good uptake in secondary regional lymph nodes if desired. Various attempts have been made to increase lymphatic uptake by changes in particle size and particle number and particle nature and these have been reviewed by Moghimi and Rajabi-Siahboomi, Prog Biophys Molec Biol 65:221–249, 1996 and Strand, Crit Rev Ther Drug Carrier Syst 6(3):211–238, 1989, incorporated herein in their entirety by reference.
While some methods have been developed for targeting and detecting specific sites in the body of a mammal, what is lacking in the prior art are effective methodologies for targeting specific lymph nodes and retaining active agents, including detection and therapeutic agents, in the targeted lymph nodes